1. Field of the Invention
The present invention relates to a method of analyzing albumin in a sample solution by mass spectrometry or liquid chromatography. More particularly, the present invention relates to a method of analyzing albumin in a sample solution, which is characterized by pre-treating the sample solution before subjecting it to mass spectrometry or liquid chromatography. Furthermore, the present invention relates to an accurate and stable method of determining the amount of, and hence the ratio of, oxidized and reduced albumin in a sample solution. Finally, the present invention relates to an albumin standard which controls the accuracy of the quantitative analysis of albumin.
2. Brief Description of the Related Art
Albumin is a protein widely and ubiquitously distributed in living bodies. Human albumin is a simple protein consisting of 585 amino acids, a molecular weight of 66 kDa, 17 disulfide bonds, and a single free cysteine per molecule. Albumin is produced in the liver and secreted into blood, and accounts for about 60% of the total protein present in plasma. Albumin is known to exhibit the following physiological effects: (1) control and maintenance of the plasma osmotic pressure, (2) transport of bilirubin, amino acids, fatty acids, hormones, metal ions, drugs, and the like, (3) a source of amino acids during malnutrition, (4) redox buffering ability, and the like. Particularly, the bond between a drug and albumin significantly affects the efficacy of the drug. As described, albumin is a protein having various functions.
Reduced albumin, oxidized albumin, glycated albumin, and the like are known to be present heterogeneously within the living body. In particular, significant albumin glycation is generally reported in diabetes (Suzuki E., Diabetes Res. 18(3), 153-158, 1992), and the amount of glycated albumin formed by glucose binding is being considered for use as a marker for monitoring blood glucose levels clinically, like hemoglobin Alc in diabetics.
However, the presence of reduced albumin (Alb(red)) and oxidized albumin (Alb(ox)) has also recently been shown to be an indicator of various diseases. For example, it has been shown that the structure of Alb(red) in the blood includes a free SH group in the 34th cysteine, as measured from the N-terminus. It has also been shown that the Alb(ox) present in the blood can add a sulfur-containing compound such as cysteine, and the like, in vivo via a disulfide bond to the SH group of the 34th cysteine (Era S, Int. J. Peptide Protein Res. 31, 435-422, 1988).
Alb(red) and Alb(ox) exist in vivo in dynamic equilibrium, as the disulfide bond reversibly forms and dissociates quickly. Accordingly, the ratio of Alb(red) to Alb(ox) in plasma indicates the redox state in blood. That is, when some oxidative stress occurs, the amount of Alb(ox) increases. Specifically, Alb(ox) is known to increase in elderly persons, patients with nephrosis syndrome, dialysis, hepatic diseases, and the like (Sogami M., J. Chromatogr., 332, 19-27, 1985; Akiharu Watanabe, Phama Medica, 19, 195-204, 2001; Suzuki E, Diabetes Res Clin Pract, 18, 153-158, 1992). In addition, diabetic patients are considered to be under oxidative stress due to increased blood oxidative products, decreased antioxidant enzyme activity, and formation of a free radical resulting in microvascular damage (Oberley L W, Free Radical Biol. Med., 5, 113-124, 1988).
When the oxidation and antioxidation reactions are not in balance and the oxidation reaction prevails, a body undergoes oxidative stress, which can be damaging. For example, cellular DNA, phospholipids on cellular membranes, proteins, and carbohydrates are damaged due to oxidative stress, resulting in advanced angiopathy, which aggravates various health conditions. As a result, oxidative stress is said to cause aging and various diseases, and it is known that substances having an antioxidant effect, such as polyphenol and the like, are good for health. Accordingly, the ability to easily monitor oxidized conditions in vivo will enable the monitoring of health conditions, and the screening for drugs and health materials.
Cirrhosis has been identified as a disease in which oxidized albumin increases. In cirrhosis patients, the blood albumin level decreases since the liver's ability to produce albumin is degraded. Preparations of human plasma albumin and branched chain amino acids are used to treat hypoalbuminemia. In hepatic diseases such as cirrhosis and the like, a decrease in the albumin level as well as an increase in oxidized albumin are observed (Watanabe A, Netrition 20, 351-357, 2004).
Moreover, fluctuation of redox balancing due to oxidative stress also occurs by impairment of renal function (Terawaki H., Kidney Int. 65(5), 1988-1993, 2004), diabetes (Suzuki E., Diabetes Res. 18(3), 153-158, 1992), rheumatism (Narazaki R., Arch. Toxicol. 14, 351-353, 1998), aging (Era S., Biochim. Biophys. Acta., 1247(1), 12-16, 1995), and the like.
In this way, albumin buffers the ability to undergo a redox reaction by forming a reductant/oxidant by itself. Accordingly, the ratio of oxidized albumin to reduced albumin is considered to reflect the redox state. Therefore, once the reduced/oxidized albumin ratio of the blood can be accurately determined in vivo, the therapeutic effect on a disease or health condition caused by oxidative stress, or the course of the disease or health condition, can be monitored.
There are several methods for determining the ratio of Alb(red) to Alb(ox), including using dye-binding to quantitatively measure the albumin. The two kinds of dye that may be used in this method are bromcresol green (BCG) and bromcresol purple (BCP). Since different reactivities of Alb(red) and Alb(ox) are obtained when using BCP, the difference in the amount of albumin quantitatively determined using BCP versus BCG indicates the ratio of Alb(ox). However, this method gives poor quanititative results and, therefore, is not reliable.
In addition, the Alb(red)-derived SH group may be quantified using a free-SH group quantitative reagent such as Ellman's Reagent, and the like (Sogami M, Int. Pept. Protein Res., 24(2), 96-103, 1984). However, this method fails to distinguish between albumin and substances with an other types of SH group.
Currently, the best method for quantifying reduced and oxidized albumin is high performance liquid chromatography (HPLC) (Sogami M., J. Chromatogr., 332, 19-27, 1985; JP-A-61-155397; JP-B-2-4863). When analyzing serum albumin using HPLC, reduced albumin (Alb(red)) and oxidized albumin (Alb(ox)) can be separately detected. The ratio of the amount of Alb(red) to the total combined amount of Alb(red) and Alb(ox) (Alb(red) %=peak area of Alb(red)/peak area of (Alb(red)+peak area of Alb(ox))×100) can be determined from the peak area ratio on the chromatogram.
However, there are some problems with HPLC, including maintaining the stability of the sample. Since reduced albumin in plasma is highly unstable, the amount of oxidized albumin increases due to natural oxidation, even when preserved at −20° C., and the amount of Alb(red) decreases. This reaction occurs in parallel with the temperature rise. Accordingly, plasma should be stored at −70° C. or below (Ryozo Muramoto, Igaku no Ayumi, 198(13), 972-976, 2001). For HPLC, the plasma stored at −70° C. or below should be thawed and immediately thereafter applied to the HPLC.
A second problem which can occur is insufficient separation of Alb(red) and Alb(ox). Since Alb(red) and Alb(ox) have only minor structural differences, it is extremely difficult to completely separate them by HPLC, and baseline separation on a chromatogram is unattainable (Keiko Yasukawa, Rinsyou Kensa, 44(8), 907-910, 2000). A third problem is that the exact structure of oxidized albumin cannot be detected. In Alb(ox), a sulfur-containing compound such as cysteine, glutathione, and the like, bonds to the 34th cysteine from the N-terminus of albumin via a disulfide bond. This structure of Alb(ox) cannot be specifically recognized using HPLC.
Recently, analysis of albumin using a mass spectrometer was reported to potentially be successful for overcoming the above-mentioned second and third problems (Keiko Yasukawa, Rinsyou Kensa, 44(8), 907-910, 2000). The advances in mass spectrometry are remarkable in recent years, and it is becoming possible to measure a protein having a large molecular weight with high accuracy and high mass resolution. Alb(ox) is heavier than Alb(red) due to the mass of the additional compound, for example, cysteine. Accordingly, Alb(red) and Alb(ox) can be separately detected by a mass spectrometer having sufficient mass resolution. In the above-mentioned literature, Yasukawa et al. measured the amount of albumin in healthy subjects and diabetic patients with an electrospray ionization mass spectrometer (ESI-MS), and was able to detect Alb(red) and Alb(ox), as well as glycated albumin.
However, the above-mentioned problem regarding the instability of reduced albumin in plasma remains a serious obstacle in the ESI-MS method. Since the ratio of oxidized albumin in plasma increases during storage above −70° C., preservation of samples is difficult. Moreover, since the amount of oxidized albumin is steadily increasing during thawing, and up to the point of measurement of the once frozen plasma, fluctuation of the measurement can occur, and inaccurate results are obtained. Accordingly, plasma kept at −70° C. or below should be thawed and immediately thereafter subjected to HPLC or ESI-MS. The time and room temperature during thawing can cause variation in the Alb(red) % value. Furthermore, substances typically used to enhance accuracy of the analysis cannot be stably preserved, therefore, control of the accuracy of the analysis is extremely difficult. In addition, automation of the analysis using an auto injector is difficult. Therefore, the measurement of the ratio of reduced albumin to oxidized albumin is not commonly practiced, but is practiced only in particular research institutions. Furthermore, while serum albumin is typically quantified by the dye-binding method, accurate quantification is problematic and therefore unavailable due to the difference in the reactions of oxidized and reduced albumins (Ryozo Muramoto, Rinsyou Kensa, 48(5), 537-544, 2004).
Generally, in analytical methods, standardized samples are important to ensure accuracy and precision. Oxidized albumin is prepared in a test tube by reacting it with a compound having a thiol group, such as cysteine, glutathione, and the like (Gabaldon M., Arch. Biochem. Biophys. 431, 178-188, 2004). However, as mentioned above, since this reaction is reversible, at oxidized albumin is easily converted to reduced albumin. Accordingly, a standardized sample which is able to maintain a constant ratio of the oxidized and reduced albumins has been desired for a long time, so that analysis can be performed with high accuracy.